Kwan et al. Journal of Neurodevelopmental Disorders (2016) 8:45 DOI 10.1186/s11689-016-9176-3

REVIEW Open Access Wnt signaling networks in spectrum disorder and Vickie Kwan, Brianna K. Unda and Karun K. Singh*

Abstract Background: Genetic factors play a major role in the risk for neurodevelopmental disorders such as autism spectrum disorders (ASDs) and intellectual disability (ID). The underlying genetic factors have become better understood in recent years due to advancements in next generation sequencing. These studies have uncovered a vast number of that are impacted by different types of mutations (e.g., de novo, missense, truncation, copy number variations). Abstract: Given the large volume of genetic data, analyzing each on its own is not a feasible approach and will take years to complete, let alone attempt to use the information to develop novel therapeutics. To make sense of independent genomic data, one approach is to determine whether multiple risk genes function in common signaling pathways that identify signaling “hubs” where risk genes converge. This approach has led to multiple pathways being implicated, such as synaptic signaling, chromatin remodeling, , and translation, among many others. In this review, we analyze recent and historical evidence indicating that multiple risk genes, including genes denoted as high-confidence and likely causal, are part of the Wingless (Wnt signaling) pathway. In the brain, Wnt signaling is an evolutionarily conserved pathway that plays an instrumental role in developing neural circuits and adult brain function. Conclusions: We will also review evidence that pharmacological therapies and genetic mouse models further identify abnormal Wnt signaling, particularly at the , as being disrupted in ASDs and contributing to disease pathology. Keywords: Autism spectrum disorders, ASD, Synapse, Wnt signaling, GSK3, Neurodevelopment, Signaling, Plasticity, Mutations, Neurotransmission, Neurogenesis, Neuronal migration

Background networks important for proper brain development. The emerging genetic landscape of Wnt signaling in ASDs While the spectrum of ASDs is reflected by the multiple ASDs and other psychiatric disorders may have heritabil- individual risk genes and loci, there is some common de- ity estimates greater than 90% [1], suggesting a strong nominator between affected individuals, which strongly genetic component to disease. With this background in suggests that disruption of the core neurodevelopmental mind, there has been an enormous advancement of new signaling pathways leads to disease symptoms. In this re- genetic technologies to discover risk-causing genes and view, we will examine accumulating evidence for the in- loci. These developments paired with an increased ability volvement of Wnt signaling in developmental cognitive to process large data sets have led to many new risk disorders. This includes emerging genetic data from genes being discovered. The number of genes and large sequencing studies, clinically used medications, chromosomal loci linked to ASDs is growing, making it and mouse models. We will also present potential ave- difficult to determine which one(s) to study. This has in- nues for therapeutic approaches, and how Wnt signaling spired the field to determine if there are links between may be modulated for treatment of patient symptoms by the genes and whether they converge into signaling leveraging clinical trial data from other fields.

* Correspondence: [email protected] Department of Biochemistry and Biomedical Sciences, Stem Cell and Cancer Research Institute, McMaster University, Hamilton, Ontario L8S 4K1, Canada

© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kwan et al. Journal of Neurodevelopmental Disorders (2016) 8:45 Page 2 of 10

Making sense of genetic findings nucleus and can act as a transcription factor that modu- It is no surprise that the clinical heterogeneity of ASDs lates the expression of target genes (Fig. 1); and (ii) can be explained, at least in part, by the large number of “non-canonical” β--independent signaling [17]. genetic mutations found through next-generation se- Interestingly, many of the in both signaling quencing. The number of mutations discovered ranges pathways localize to the synapse and play important in the several hundreds, according to well-known functions in synaptic growth and maturation [18–23]. sources such as the Simons Foundation Autism Research There are now multiple lines of evidence implicating this Initiative (SFARI). There is much difficulty in trying to pathway in the etiology and pathophysiology of ASD and understand the biological etiology of ASDs when so intellectual disability (ID). While the human genetic data many genes are involved. One hypothesis is that the gen- is an important supporting factor, it is not the only one. etic lesions disrupt specific signaling pathways during There are a number of mouse genetic knockout (KO) discrete time points of brain development. For example, models targeting Wnt signaling molecules, describing many of the initial genetic studies identified genes in- molecular, cellular, electrophysiological, and behavioral volved in synapse development and refinement. This was deficits that are consistent with ASD and ID. Further- largely based on findings that multiple genes in syn- more, the genes involved in Wnt signaling are of signifi- dromic forms of ASDs (e.g., Fragile X syndrome (FMR1), cant clinical interest because there are a variety of Rett syndrome (MECP2), Angelman syndrome (UBE3A), approved drugs that either inhibit or stimulate this and genes that cause non-syndromic forms of ASDs pathway. (e.g., the Shank and neuroligin/neurexin family of pro- teins) have important roles during synapse development Genetic evidence implicating Wnt signaling genes and and refinement [2–8]. This suggests that the disruption support by cellular models of postnatal synaptic maturation could increase the risk CHD8 for developing ASDs and related disorders. However, The strongest single candidate gene for non-syndromic there has been accumulating evidence that other brain ASDs is chromodomain helicase DNA binding protein 8 developmental milestones are also vulnerable, such as (CHD8) [24–30]. There are multiple de novo, truncating, prenatal brain development (e.g., neurogenesis) [9], or or missense mutations discovered in CHD8 in individ- postnatal development of non-neuronal cells (e.g., oligo- uals with ASDs [27–29, 31–34]. CHD8 is found at active dendrocytes during myelination and microglia function) transcription sites with histone modifications H3K4me3 [10–13]. This is also supported by the implication of or H3K27ac, and it is thought to directly activate genes discrete cell types in the brain based on the identifica- by binding near the transcriptional start site and pro- tion of specific risk genes expressed in those cells (e.g., moting transcription factor activity or recruitment. It inhibitory ) [14–16]. In the current review, we can also indirectly impact transcription by interacting take an alternative approach that is not in contrast to with modified histone sites and other co-regulators to these hypotheses but examines whether there is conver- make chromatin more assessable [24, 34–36]. Interest- gence of multiple risk genes onto specific signaling path- ingly, one of the major pathways regulated by CHD8 is ways, which ultimately impact multiple cell types and/or canonical Wnt signaling [37, 38]. Previous work charac- developmental processes. We put forth the notion that terized CHD8 as a negative regulator of canonical Wnt by focusing on a specific pathway and dissecting which signaling, which fits with the hypothesis that elevated ca- molecular players in that pathway are important for dis- nonical Wnt signaling activity causes excessive prolifera- ease pathophysiology, it may offer an opportunity to tion of embryonic neural progenitor cells (NPCs) in the identify key proteins to be pharmacologically targeted by brain and may in part explain the macrocephaly (“big drug therapies to treat these disorders. brain”) phenotype observed in patients [27]. Further- more, recent studies in human neural progenitors lack- Review ing one copy of CHD8 support this notion, as it revealed Convergent evidence for Wnt signaling many target genes controlled by CHD8 that are involved One pathway highlighted in the multitude of genetic in the regulation of spine head size [34, 39, 40]. However studies is the Wingless (Wnt) signaling. This pathway is a recent study discovered that CHD8 is in fact a positive highly studied and conserved from lower to higher or- regulator of Wnt/β-catenin signaling NPCs, while simul- ganisms, where it plays a variety of roles in almost all taneously demonstrating that it negatively regulates the tissues. Broadly speaking, Wnt signaling in the brain can pathway in non-neuronal cell lines [41]. Given this unex- be divided into two main pathways: (i) “canonical” sig- pected finding, this suggests that CHD8 regulates Wnt naling that results in the stabilization of the protein β- signaling in a cell-specific manner, and the possibility catenin (encoded by CTNNB1), which upon stabilization, that some of the CHD8 mutations may not be as simple can exert functions at the plasma membrane or in the as loss-of-function for Wnt signaling. Further work is Kwan et al. Journal of Neurodevelopmental Disorders (2016) 8:45 Page 3 of 10

Fig. 1 Summary of genetic and pharmacological evidence implicating Wnt signaling in developmental cognitive disorders. This diagram depicts the canonical Wnt signaling pathway which consists of Wnt binding to the Frizzled-LRP5/6 co-receptor complex and inhibiting the disassembly of the destruction complex which results in stabilized β-catenin levels, translocation to the nucleus, and initiation of Wnt-dependent gene transcription. Several genes in the canonical Wnt pathway are also identified as high-risk genes associated with autism and intellectual disability (CHD8, DDX3X,andTCF4). Furthermore, listed are a number of pharmacological agents and drugs that target Wnt signaling molecules and can modulate the pathway. Many of the genes listed are shared between different upstream ligand-receptor pathways needed to clarify how CHD8 regulates Wnt signaling in catenin exists in a protein network, including CHD8 and different cell types in the brain, and how patients with other ASD or ID associated genes [25]. The relationship CHD8 mutations acquire macrocephaly. It is also im- between Wnt signaling and chromatin remodeling fac- portant to note that Wnt signaling is only one neurode- tors demonstrates that proper interplay between these velopmental pathway regulated by CHD8, and recent pathways is important for appropriate levels of canonical studies have identified many others (e.g., chromatin re- Wnt-dependent gene transcription. CHD8 regulates β- modeling). Therefore, future work needs to determine catenin-mediated canonical Wnt signaling, which could the precise mechanisms and time points during which occur by CHD8 directly by binding to β-catenin or indir- CHD8 regulates Wnt signaling during neurodevelop- ectly by inhibiting the recruitment of co-factors required ment. This is important to better comprehend how pre- for transcription at promoter sequences. Future work natal brain development could be compromised in will need to determine precisely which neural progenitor individuals with CHD8 mutations. sub-populations are most sensitive to disruptions in ca- nonical Wnt/β-catenin signaling during prenatal brain CTNNB1 (β-catenin) development. For example, previous studies indicated β-catenin is a central player in the canonical Wnt signal- that global stabilization of β-catenin in the cortex, which ing pathway and works with co-factors to initiate Wnt- elevates canonical Wnt signaling, leads to brain over- dependent gene transcription (Fig. 1). It has directly growth due to increased cycling neural progenitor cells been implicated in ASDs due to the identification of de and production of neurons [43]. In contrast, deletion of novo mutations in the CTNNB1 gene in patients with β-catenin from parvalbumin-expressing inhibitory neu- ASD using exome sequencing [25, 28, 29, 42]. Given the rons leads to ASD-like defects in neuronal activation core nature of this gene in canonical Wnt signaling, this and behavior, such as social interaction and object rec- strongly places aberrations in Wnt signaling as one of ognition impairments [44]. the main networks in ASD pathogenesis. Network ana- β-caxtenin also functions in pathways other than ca- lysis from data also indicates that β- nonical Wnt signaling, such as cell adhesion at the Kwan et al. Journal of Neurodevelopmental Disorders (2016) 8:45 Page 4 of 10

plasma membrane. β-catenin interacts with cadherins to TCF7L2 (TCF4) regulate dendritic spine growth and synaptic competition Recent studies have identified mutations in CTNNB1 during the process of postnatal period of dendritic prun- within individuals with developmental delay and ID who ing [45]. A recent study identified that dominant muta- do not have an ASD [46, 61, 62]. Additional support tions in CTNNB1 from ID patients when expressed in comes from another ASD exome sequencing study that mice have a reduced affinity for membrane-associated identified de novo loss of function variants in TCF7L2 cadherins [46]. This is associated with a decrease in cad- (transcription factor 7-like 2 (T-cell specific, HMG-box)) herin interaction and decreased intrahemispheric connec- [63, 64], which also goes by the name TCF4, and is con- tions, with deficits in dendritic branching, long-term fused with transcription factor 4 (TCF4) as they share potentiation, and cognitive function [46]. Therefore, it is the same symbol. Importantly, TCF7L2 is a key player in possible that mutations in β-catenin could lead to aberrant canonical Wnt signaling as it helps to initiate gene tran- Wnt signaling developmentally, concurrent with disrup- scriptional response when Wnt ligands bind their recep- tion of plasma membrane signaling at synaptic sites, both tors on the membrane, and the signal is transduced to leading to disrupted gene transcription programs and ab- the nucleus (Fig. 1). These genetic studies directly impli- normal synaptic plasticity. Furthermore, both of these cate de novo missense variants in TCF4 in ASD, suggest- pathogenic events would increase disease risk. In this re- ing that perturbations to the core canonical signaling gard, these pathways are also likely connected given that complex play a pathogenic role. However, the role of β-catenin signaling at the membrane impacts nuclear TCF4 in brain development and which time points and Wnt-dependent transcription [47]. cell types it regulates is not well known and needs to be identified in future studies.

PTEN DDX3X Another high-risk autism candidate gene that has A recent study identified de novo mutations in DEAD- roles in Wnt signaling is PTEN due to the discovery box helicase 3, X-linked (DDX3X) in a population of un- of numerous individuals with mutations [29, 48–50]. explained ID [65]. DDX3X was identified as a regulator PTEN has multiple functions but is best known for of the Wnt-β-catenin network, via regulation of the kin- its role as a negative regulator of the PI3K-Akt- ase activity of CK1ε, to promote phosphorylation of Dvl, mTOR pathway. Individuals with heterozygous muta- both critical factors in canonical Wnt signaling (Fig. 1). tions in PTEN are also at risk for macrocephaly, in- Moreover, in model systems testing the Wnt pathway, dicating that PTEN regulates brain size, which is DDX3X was found to be required for Wnt-β-catenin sig- thought to be an impact on certain individuals with naling in mammalian cells through loss of function stud- ASD [51–59]. Multiple mouse models of Pten have ies [66]. Therefore, in addition to ASD, Wnt-β-catenin further strengthened the notion that it is an import- signaling may be disrupted in generalized ID and devel- ant regulator of different neural circuits associated opmental disorders (DD), further demonstrating the im- with ASDs and cognition. For example, an early study portance of this pathway in proper neurodevelopment. identified that global Pten +/- mice have impairments However, a critical question that remains is how do mu- in social interaction behaviors [52]. Knocking out tations in Wnt signaling molecules give rise to different Pten in the cerebellar Purkinje cells led to impaired phenotypes, such as ASD versus ID/DD? This insight sociability, repetitive behavior, and deficits in motor would provide tremendous clinical utility, as it will allow learning. Additionally, knocking out Pten in a subset caregivers and clinicians to plan therapies according to of cortical excitatory neurons results in profound the future outcome. synaptic signaling changes [60], while ASD-associated Pten alleles expressed in inhibitory neurons cause Animal models support the involvement of Wnt signaling excitatory/inhibitory imbalances [54]. Interestingly, Mouse models provide support for Wnt signaling as a PTEN has recently been identified to function with clinically relevant pathway for developmental cognitive β-catenin to regulate normal brain growth, implicating disorders. First, modeling of high-risk (i.e., causative) PTEN in Wnt signaling. It was discovered that ASD genes, for example using gene knockouts (KO), β-catenin signaling is elevated in a mouse model of offer the opportunity to determine the neural circuits Pten (Pten +/-), and a heterozygous mutation in β-catenin and brain regions responsible for causing ASD-like suppresses the excessive cortical brain growth in Pten +/- behavior. Second, interrogation of other genes in the mice [53]. This indicates that multiple ASD risk factors same pathway, which are not directly involved in ASD likely converge upon neural progenitor proliferation dur- from human genetic studies, offers the opportunity to ing embryonic brain development, potentially through further support that pathway in ASD pathophysiology. regulation of canonical Wnt signaling. For example, while no ASD-specific genetic mutations Kwan et al. Journal of Neurodevelopmental Disorders (2016) 8:45 Page 5 of 10

have been identified in the disheveled genes (Dvl 1, 2 that DISC1 positively regulates canonical Wnt signaling and 3), Dvl1 or Dvl1/3 KO mice display adult social and [85], which has been followed up by other studies [86– repetitive behavioral abnormalities, which are the core 89]. Several follow-up studies on multiple mouse models features of ASD symptoms [67–69]. This type of ex- of Disc1 demonstrate that DISC1 perturbation causes ample lends further evidence that perturbation of the significant neurodevelopmental phenotypes, including core Wnt signaling transduction molecules like Dvl1/3 cognitive defects and psychiatric-like behavioral manifes- can result in ASD-like abnormalities even though they tations [90–94]. are not directly implicated in human genetic studies. There are other known regulators of Wnt signaling In addition to Dvl1/3, recent studies have highlighted that when disrupted leads to neurocognitive and neuro- that conditional or complete KO mouse models of other developmental phenotypes. A recent example is Ankyr- genes involved in Wnt signaling support the pathway be- inG (Ank3), which was found to possess a genome-wide ing involved in ASD-like phenotypes. One of the best- significant signal in [95, 96]. Ank3 is a studied genes is glycogen synthase kinase 3 (GSK3) α and scaffolding protein that localizes to the nodes of Ranvier β, which is a negative regulator of canonical Wnt signal- in mature neurons, important for the formation and ing and also plays important roles directly at the synapse maintenance of the initial segment [95]. It has also (Fig. 1). It is well-established that the inhibition of GSK3 been shown to regulate glutamatergic synapse structure using lithium or specific inhibitors (e.g., CHIR 99021) and function through modulation of AMPAR-mediated causes an increase in activation of the canonical synaptic transmission and maintenance of dendritic transcriptional pathway of Wnt signaling [70]. Gsk-3β spine morphology [97]. Interestingly, Ank3 is a negative heterozygous (+/-) mice display behaviors that resemble regulator of canonical Wnt signaling during embryonic wild type (WT) mice treated with lithium, a drug that is neurogenesis in the mouse brain and functionally inter- used to treat bipolar disorder [71], demonstrating that acts with DISC1 to regulate this process [46]. Ank3 het- disruption of Wnt signaling leads to behavioral abnor- erozygous mice possess behavioral phenotypes such as malities. Furthermore, forebrain-specific deletion of Gsk- reduced anxiety and increased motivation for reward, 3β in excitatory neurons leads to anxiolytic and pro- which can be corrected by modulating Wnt signaling social effects [72], suggesting that GSK-3β plays import- through GSK-3β [98], demonstrating the clinical involve- ant roles in normal behavior. The most convincing evi- ment of this pathway. In addition to Ank3, other recently dence that GSK3 and Wnt signaling may be involved in characterized genes in mice also support a role for Wnt developmental cognitive disorders is its role in Fragile X signaling in neurodevelopmental disorders. DIX domain syndrome (FXS), which is the most commonly inherited containing 1 (DIXDC1) is a positive regulator of Wnt form of intellectual disability and is linked to ASDs [73, signaling and neurogenesis through binding to DISC1 74]. Fragile X Mental Retardation Protein (FMRP) KO [99] and a Dixdc1 KO mouse displayed behaviors associ- mice, which is a FXS model, has been shown to possess ated with neuropsychiatric disorders such as abnormal a dysregulation of GSK3β activity. Specifically, GSK-3β startle reflex and reduced social interaction [100]. The protein and its activity is pathogenically elevated in FXS behavioral phenotypes displayed by these mice could be models [75, 76], and pharmacological correction of this rescued through lithium or GSK3 inhibitor treatment enhanced activity using lithium or GSK3 inhibitors in [101, 102]. mice rescues neurobehavioral and brain morphological There are three other Wnt signaling-related genes that abnormalities [77–82]. Furthermore, studies investigat- have been characterized in mice that lend further sup- ing FXS mice demonstrated that Wnt signaling is also port to the involvement of Wnt signaling in develop- disrupted [83, 84]. Of course, GSK-3β has many down- mental cognitive disorders (see Fig. 1). The first is stream signaling targets, one of which is the Wnt signal- adenomatous polyposis coli (APC), which is a critical ing pathway; demonstrating that modulation of GSK-3β component of the destruction complex in the canonical activity can have therapeutic effects beyond the treat- Wnt pathway and is important for neural plasticity, ment of bipolar disorder. learning, and memory in mice. A conditional Apc KO Another well-studied gene in relation to ASD and psy- mouse showed increased synaptic spine density, elevated chiatric disorders is disrupted in 1 frequency of miniature excitatory postsynaptic potentials (DISC1). While the genetic evidence linking DISC1 to (mEPSPs), enhanced long-term potentiation (LTP), and developmental cognitive disorders is not strong, the ASD-like behaviors (e.g., repetitive behaviors and re- multiple cellular and mouse models of Disc1 perturb- duced social interest) [103]. Second, analysis of a ation has led to important findings linking Wnt signaling Prickle2 mouse model demonstrates its importance in to abnormal neurodevelopment. For example, a land- ASD-related neural circuits and behavior [104, 105]. mark study initially described DISC1 as an inhibitor of Prickle2 is a postsynaptic protein that interacts with GSK-3β, similar to the actions of lithium, demonstrating PSD-95 and is part of the non-canonical Wnt signaling Kwan et al. Journal of Neurodevelopmental Disorders (2016) 8:45 Page 6 of 10

pathway [106]. The Prickle2 KO mouse has previously changes to canonical Wnt signaling during prenatal been shown to be more sensitive to seizures and also brain development can have a profound impact on brain shows reduced dendrite branching, synapse number, and size and function. These results suggest a causal rela- (PSD) size, as well as behavioral ab- tionship between abnormal modulation of Wnt signaling normalities (learning abnormalities, altered social inter- during neurodevelopment and autism-like features [115]. action, and behavioral inflexibility). Although the involvement of Prickle2 implicates non-canonical Wnt Hope for ASD treatment using Wnt signaling modulators? signaling, the phenotypes associated with the KO mouse There is only one FDA approved for ASDs, which is demonstrate that multiple aspects of Wnt signaling (ca- used to treat irritability associated with ASDs (risperi- nonical and non-canonical) are important for the estab- done, an antipsychotic medication), demonstrating the lishment of neural circuits that are disrupted in ASD. urgent need to find new medications. Many of the medi- While the studies on APC and Prickle2 do not directly cations used are “off-label” (e.g., antidepressants, anti- implicate abnormal Wnt signaling, we speculate that convulsants, stimulants, and antianxiety medications) these mice would have alterations in this pathway due to and do not treat the core symptoms, and can have very the importance of these molecules in Wnt signaling in strong side effects. While these medications have mul- neural cells. Third, a recent study identified that rare tiple modes of molecular action, interestingly, many im- missense variants in the Wnt1 gene discovered in ASD pact Wnt signaling. For example, haloperidol (typical patients show abnormal activation of the Wnt signaling antipsychotic medication) is known to inhibit dopamine pathway, providing evidence that subtle changes to the receptors, thereby increasing GSK3β inhibition through coding sequence of Wnt signaling molecules alter bio- Akt activation [116], which impacts downstream canon- logical signaling [107]. Together, these studies indicate ical Wnt signaling [117, 118]. Selective serotonin re- that when analyzed using animal models, members of uptake inhibitors (SSRIs) (e.g., fluoxetine), which are the Wnt signaling pathway, which have no link to dis- used to treat depression, potentially by increasing hippo- ease from human genetic studies, demonstrate how dis- campal neurogenesis in mice, have been shown to ruption of this core signaling pathway in the brain antagonize canonical Wnt signaling, which causes a re- results in developmental phenotypes consistent with hu- duction in expression of the serotonin transporter man disease. (SERT) in serotonergic raphe neurons through miR-16 [119, 120]. Additionally, lithium is a well-known treat- Targeting Wnt signaling in ASD/ID mouse models ment for bipolar disorder, and one of its main activities In addition to animal models, there are two specific is inhibition of GSK-3β, which positively stimulates the drug-induced models that implicate Wnt signaling. The canonical Wnt pathway [121–123]. Stimulants such as first is valproic acid (VPA), which is thought to increase Methylphenidate (e.g., Ritalin) can function as a negative the risk for ASD through exposure to a pregnant woman regulator of the canonical pathway by activating GSK-3β during prenatal development [108]. The administration [124, 125]. In this regard, various GSK-3β inhibitors have of VPA to pregnant mice has long been used as a model been used to rescue neurogenesis defects in mouse of ASD, as the offspring of these mice develop ASD-like models of psychiatric disorders and ASD, which also deficits in brain structure, neuronal signaling, and be- stimulate canonical Wnt signaling pathways [69]. Taken havior [109]. VPA has many targets, but one of its better together, while it is important to be cautious of the mul- characterized effects is the stimulation of the canonical tiple mechanisms of action of all of the classes of medi- Wnt signaling pathway through modulation of histone cations discussed here, it is intriguing to find that all of deacetylase and GSK3 [110–114], demonstrating that ab- them either directly or indirectly impact canonical Wnt normal Wnt signaling likely plays an important role in signaling in the brain to some degree. This suggests that the pathogenicity of VPA. A second model that was re- abnormal Wnt signaling likely plays a core role in the cently developed and more specifically implicates Wnt disease pathogenesis of developmental cognitive disor- signaling is exposure of pregnant mice to the compound, ders, and restoring normal levels of this pathway with XAV939, which is a tankyrase inhibitor, resulting in en- medications could be an option for treatment. hanced canonical Wnt signaling [115]. This leads to the Many times the medications tested in pilot clinical tri- expansion of the intermediate progenitor cell population als for neurological disorders are failed drugs from can- in the developing cerebral cortex. The result of exposure cer trials. There are many drugs developed and tested as to XAV939 is an overpopulation of neurons in the cor- modulators of Wnt signaling in the cancer field that tex, which disrupts the development and function of could potentially be repurposed for developmental cog- dendrites and dendritic spines of excitatory neurons and nitive disorders. In cases where a reduction in Wnt sig- alters the distribution of interneurons. These mice ex- naling is thought to underlie the pathology of the hibit ASD-like behavioral abnormalities, implicating that disorder, usage of compounds that elevated canonical Kwan et al. Journal of Neurodevelopmental Disorders (2016) 8:45 Page 7 of 10

Wnt signaling could be applied. An example of this is CIHR, Scottish Rite Charitable Foundation, NARSAD, Brain Canada, and the JP GSK-3β inhibitors that have failed in cancer trials but Bickell Foundation. may be effective for ASDs and ID (e.g., Tideglusig, Clini- Funding calTrials.gov identifier: NCT02586935). In cases where Financial support for this manuscript was provided by grants from the elevated Wnt signaling is thought to contribute to dis- following agency to Karun Singh: the Natural Sciences and Engineering Research Council, Scottish Rite Charitable Foundation of Canada, JP Bickell ease pathology, there are many potential options to in- Medical Foundation, Brain Canada, and the Brain and Behavior Research hibit canonical Wnt signaling using chemicals (Fig. 1) Foundation. that inhibit the interaction between β-catenin and its targets (e.g., inhibiting β-catenin interaction with the Availability of data and materials Not applicable. TCF factors), disheveled inhibitors (through targeting of the PDZ domain which generally inhibit the Frizzled– Authors’ contributions PDZ interaction), and tankyrase inhibitors (e.g., The topics of discussion were generated by KKS and VK, and BU assisted KKS in writing the manuscript. VK designed the figures with KKS. All authors read XAV939, which induces the stabilization of axin by inhi- and approved the final manuscript. biting the poly (ADP)-ribosylating enzymes tankyrase 1 and tankyrase 2) [126]. These candidate compounds Competing interests The authors declare that they have no competing interests. may be of clinical use in cases where it is thought that the genetic risk factor for ASD or ID causes elevated ca- Consent for publication nonical Wnt signaling (e.g., potentially some individuals Not applicable. with CHD8 mutations); however, even if these drugs Ethics approval made it to the clinic, they would likely have to be deliv- Not applicable. ered in utero, since embryonic brain development is most affected by such genetic mutations, posing ethical Received: 30 June 2016 Accepted: 7 November 2016 issues for pre-diagnosis therapies. References 1. Lichtenstein P, Carlstrom E, Rastam M, Gillberg C, Anckarsater H. The Conclusions genetics of autism spectrum disorders and related neuropsychiatric The goal towards better understanding and treating de- disorders in childhood. Am J Psychiatry. 2010;167(11):1357–63. 2. Ebrahimi-Fakhari D, Sahin M. Autism and the synapse: emerging velopmental cognitive disorders is a difficult road and mechanisms and mechanism-based therapies. Curr Opin Neurol. 2015;28(2): will require a multifaceted research and clinical ap- 91–102. proach to be successful. In this review, we present evi- 3. Shepherd GM, Katz DM. Synaptic microcircuit dysfunction in genetic models of neurodevelopmental disorders: focus on Mecp2 and Met. Curr Opin dence that one such signaling pathway that may be Neurobiol. 2011;21(6):827–33. central to disease pathogenesis and treatment is the Wnt 4. Mullins C, Fishell G, Tsien RW. Unifying views of autism spectrum signaling network. Ongoing and future genetic studies disorders: a consideration of autoregulatory feedback loops. . 2016;89(6):1131–56. will need to determine the strength of association of this 5. Habela CW, Song H, Ming GL. Modeling synaptogenesis in schizophrenia pathway with disease; however, given the medications and autism using human iPSC derived neurons. Mol Cell Neurosci. 2016;73: and drugs that target this pathway currently available, 52–62. 6. Huber KM, Klann E, Costa-Mattioli M, Zukin RS. Dysregulation of mammalian this presents an opportunity for new clinical trials in the target of rapamycin signaling in mouse models of autism. J Neurosci. 2015; near future. 35(41):13836–42. 7. Bourgeron T. From the genetic architecture to synaptic plasticity in autism spectrum disorder. Nat Rev Neurosci. 2015;16(9):551–63. Abbreviations 8. Volk L, Chiu SL, Sharma K, Huganir RL. Glutamate in human ANK3: AnkyrinG; APC: Adenomatous polyposis coli; ASD: Autism spectrum cognitive disorders. Annu Rev Neurosci. 2015;38:127–49. disorder; BP: Bipolar disorder; CHD8: Chromatin-helicase-DNA-binding protein 9. Packer A. Neocortical neurogenesis and the etiology of autism spectrum 8; CIHR: Chiron; CTNNB1: (β-catenin); DDX3X: DEAD-box helicase 3, X-linked; disorder. Neurosci Biobehav Rev. 2016;64:185–95. DISC1: Disrupted in schizophrenia 1; DIXDC1: Dix domain containing 1; 10. Pacey LK, Xuan IC, Guan S, Sussman D, Henkelman RM, Chen Y, Thomsen C, DVL: Disheveled; FDA: Food and drug administration; FMR1: Fragile X mental Hampson DR. Delayed myelination in a mouse model of fragile X retardation 1; FMRP: Fragile X mental retardation protein; FXS: Fragile X syndrome. Hum Mol Genet. 2013;22(19):3920–30. syndrome; GSK: Glycogen synthase kinase; ID: Intellectual disability; 11. Ameis SH, Catani M. Altered white matter connectivity as a neural substrate KO: Knockout; LTP: Long-term potentiation; MECP2: Methyl CpG binding for social impairment in Autism Spectrum Disorder. Cortex. 2015;62:158–81. protein 2; miniature excitatory postsynaptic potentials; mEPSPs: Miniature 12. Estes ML, McAllister AK. Immune mediators in the brain and peripheral excitatory postsynaptic potentials; NDD: Neurodevelopmental disorders; tissues in autism spectrum disorder. Nat Rev Neurosci. 2015;16(8):469–86. Prickle2: Prickle planar cell polarity protein 2; PSD: Postsynaptic density; 13. Bilimoria PM, Stevens B. Microglia function during brain development: new PTEN: Phosphatase and tensin homolog; SERT: Serotonin transporter; insights from animal models. Brain Res. 2015;1617:7–17. SFARI: Simons Foundation Autism Research Initiative; SSRI: Selective 14. Gao R, Penzes P. Common mechanisms of excitatory and inhibitory serotonin reuptake inhibitor; UBE3A: Ubiquitin-protein ligase E3A; imbalance in schizophrenia and autism spectrum disorders. Curr Mol Med. VPA: Valproic acid; Wnt: Wingless; WT: Wild type 2015;15(2):146–67. 15. Marin O. Interneuron dysfunction in psychiatric disorders. Nat Rev Neurosci. Acknowledgements 2012;13(2):107–20. We would like to acknowledge the following agencies for funding support 16. Nelson SB, Valakh V. Excitatory/inhibitory balance and circuit homeostasis in for research in the laboratory on the topics discussed in this review: NSERC, autism spectrum disorders. Neuron. 2015;87(4):684–98. Kwan et al. Journal of Neurodevelopmental Disorders (2016) 8:45 Page 8 of 10

17. Salinas PC, Zou Y. Wnt signaling in neural circuit assembly. Annu Rev 41. Durak O, Gao F, Kaeser-Woo YJ, Rueda R, Martorell AJ, Nott A, Liu CY, Neurosci. 2008;31:339–58. Watson LA, Tsai LH. Chd8 mediates cortical neurogenesis via transcriptional 18. Caracci MO, Avila ME, De Ferrari GV. Synaptic Wnt/GSK3beta signaling hub regulation of cell cycle and Wnt signaling. Nat Neurosci. 2016. in autism. Neural Plast. 2016;2016:9603751. 42. Sanders SJ, Murtha MT, Gupta AR, Murdoch JD, Raubeson MJ, Willsey AJ, 19. Purro SA, Galli S, Salinas PC. Dysfunction of Wnt signaling and synaptic Ercan-Sencicek AG, DiLullo NM, Parikshak NN, Stein JL, et al. De novo disassembly in neurodegenerative diseases. J Mol Cell Biol. 2014;6(1):75–80. mutations revealed by whole-exome sequencing are strongly associated 20. Oliva CA, Vargas JY, Inestrosa NC. Wnts in adult brain: from synaptic with autism. Nature. 2012;485(7397):237–41. plasticity to cognitive deficiencies. Front Cell Neurosci. 2013;7:224. 43. Chenn A, Walsh CA. Regulation of cerebral cortical size by control of cell 21. Stamatakou E, Salinas PC. Postsynaptic assembly: a role for Wnt signaling. cycle exit in neural precursors. Science. 2002;297(5580):365–9. Dev Neurobiol. 2014;74(8):818–27. 44. Dong F, Jiang J, McSweeney C, Zou D, Liu L, Mao Y. Deletion of CTNNB1 in 22. Budnik V, Salinas PC. Wnt signaling during synaptic development and inhibitory circuitry contributes to autism-associated behavioral defects. Hum plasticity. Curr Opin Neurobiol. 2011;21(1):151–9. Mol Genet. 2016. Epub ahead of print. 23. Okerlund ND, Cheyette BN. Synaptic Wnt signaling—a contributor to major 45. Bian WJ, Miao WY, He SJ, Qiu Z, Yu X. Coordinated spine pruning and psychiatric disorders? J Neurodev Disord. 2011;3(2):162–74. maturation mediated by inter-spine competition for cadherin/catenin 24. Barnard RA, Pomaville MB, O’Roak BJ. Mutations and modeling of the complexes. Cell. 2015;162(4):808–22. chromatin remodeler CHD8 define an emerging autism etiology. Front 46. Tucci V, Kleefstra T, Hardy A, Heise I, Maggi S, Willemsen MH, Hilton H, Neurosci. 2015;9:477. Esapa C, Simon M, Buenavista MT, et al. Dominant beta-catenin mutations 25. Krumm N, O’Roak BJ, Shendure J, Eichler EE. A de novo convergence of autism cause intellectual disability with recognizable syndromic features. J Clin genetics and molecular neuroscience. Trends Neurosci. 2014;37(2):95–105. Invest. 2014;124(4):1468–82. 26. Sanders SJ. First glimpses of the neurobiology of autism spectrum disorder. 47. Zhang J, Shemezis JR, McQuinn ER, Wang J, Sverdlov M, Chenn A. AKT Curr Opin Genet Dev. 2015;33:80–92. activation by N-cadherin regulates beta-catenin signaling and neuronal 27. Bernier R, Golzio C, Xiong B, Stessman HA, Coe BP, Penn O, Witherspoon K, differentiation during cortical development. Neural Dev. 2013;8:7. Gerdts J, Baker C, Vulto-van Silfhout AT, et al. Disruptive CHD8 mutations 48. Spinelli L, Black FM, Berg JN, Eickholt BJ, Leslie NR. Functionally distinct define a subtype of autism early in development. Cell. 2014;158(2):263–76. groups of inherited PTEN mutations in autism and tumour syndromes. J 28. O’Roak BJ, Vives L, Girirajan S, Karakoc E, Krumm N, Coe BP, Levy R, Ko A, Med Genet. 2015;52(2):128–34. Lee C, Smith JD, et al. Sporadic autism exomes reveal a highly 49. Frazier TW, Embacher R, Tilot AK, Koenig K, Mester J, Eng C. Molecular and interconnected protein network of de novo mutations. Nature. 2012; phenotypic abnormalities in individuals with germline heterozygous PTEN 485(7397):246–50. mutations and autism. Mol Psychiatry. 2015;20(9):1132–8. 29. O’Roak BJ, Vives L, Fu W, Egertson JD, Stanaway IB, Phelps IG, Carvill G, 50. McBride KL, Varga EA, Pastore MT, Prior TW, Manickam K, Atkin JF, Herman Kumar A, Lee C, Ankenman K, et al. Multiplex targeted sequencing identifies GE. Confirmation study of PTEN mutations among individuals with autism recurrently mutated genes in autism spectrum disorders. Science. 2012; or developmental delays/mental retardation and macrocephaly. Autism Res. 338(6114):1619–22. 2010;3(3):137–41. 30. Krumm N, Turner TN, Baker C, Vives L, Mohajeri K, Witherspoon K, Raja A, 51. Page DT, Kuti OJ, Prestia C, Sur M. Haploinsufficiency for Pten and Serotonin Coe BP, Stessman HA, He ZX, et al. Excess of rare, inherited truncating transporter cooperatively influences brain size and social behavior. Proc Natl mutations in autism. Nat Genet. 2015;47(6):582–8. Acad Sci U S A. 2009;106(6):1989–94. 31. Neale BM, Kou Y, Liu L, Ma’ayan A, Samocha KE, Sabo A, Lin CF, Stevens C, 52. Kwon CH, Luikart BW, Powell CM, Zhou J, Matheny SA, Zhang W, Li Y, Baker Wang LS, Makarov V, et al. Patterns and rates of exonic de novo mutations SJ, Parada LF. Pten regulates neuronal arborization and social interaction in in autism spectrum disorders. Nature. 2012;485(7397):242–5. mice. Neuron. 2006;50(3):377–88. 32. Talkowski ME, Rosenfeld JA, Blumenthal I, Pillalamarri V, Chiang C, Heilbut A, 53. Chen Y, Huang WC, Sejourne J, Clipperton-Allen AE, Page DT. Pten Ernst C, Hanscom C, Rossin E, Lindgren AM, et al. Sequencing chromosomal mutations alter brain growth trajectory and allocation of cell types through abnormalities reveals neurodevelopmental loci that confer risk across elevated beta-catenin signaling. J Neurosci. 2015;35(28):10252–67. diagnostic boundaries. Cell. 2012;149(3):525–37. 54. Vogt D, Cho KK, Lee AT, Sohal VS, Rubenstein JL. The parvalbumin/ 33. McCarthy SE, Gillis J, Kramer M, Lihm J, Yoon S, Berstein Y, Mistry M, Pavlidis somatostatin ratio is increased in Pten mutant mice and by human PTEN P, Solomon R, Ghiban E, et al. De novo mutations in schizophrenia implicate ASD alleles. Cell Rep. 2015;11(6):944–56. chromatin remodeling and support a genetic overlap with autism and 55. Clipperton-Allen AE, Page DT. Decreased aggression and increased intellectual disability. Mol Psychiatry. 2014;19(6):652–8. repetitive behavior in Pten haploinsufficient mice. Genes Brain Behav. 2015; 34. Sugathan A, Biagioli M, Golzio C, Erdin S, Blumenthal I, Manavalan P, 14(2):145–57. Ragavendran A, Brand H, Lucente D, Miles J, et al. CHD8 regulates 56. Clipperton-Allen AE, Page DT. Pten haploinsufficient mice show broad brain neurodevelopmental pathways associated with autism spectrum disorder in overgrowth but selective impairments in autism-relevant behavioral tests. neural progenitors. Proc Natl Acad Sci U S A. 2014;111(42):E4468–77. Hum Mol Genet. 2014;23(13):3490–505. 35. Wilkinson B, Grepo N, Thompson BL, Kim J, Wang K, Evgrafov OV, Lu W, 57. Takeuchi K, Gertner MJ, Zhou J, Parada LF, Bennett MV, Zukin RS. Knowles JA, Campbell DB. The autism-associated gene chromodomain Dysregulation of synaptic plasticity precedes appearance of morphological helicase DNA-binding protein 8 (CHD8) regulates noncoding and defects in a Pten conditional knockout mouse model of autism. Proc Natl autism-related genes. Transl Psychiatry. 2015;5:e568. Acad Sci U S A. 2013;110(12):4738–43. 36. Cotney J, Muhle RA, Sanders SJ, Liu L, Willsey AJ, Niu W, Liu W, Klei L, Lei J, Yin 58. Tilot AK, Frazier 2nd TW, Eng C. Balancing proliferation and connectivity in PTEN- J, et al. The autism-associated chromatin modifier CHD8 regulates other autism associated autism spectrum disorder. Neurotherapeutics. 2015;12(3):609–19. risk genes during human neurodevelopment. Nat Commun. 2015;6:6404. 59. Zhou J, Parada LF. PTEN signaling in autism spectrum disorders. Curr Opin 37. Thompson BA, Tremblay V, Lin G, Bochar DA. CHD8 is an ATP-dependent Neurobiol. 2012;22(5):873–9. chromatin remodeling factor that regulates beta-catenin target genes. Mol 60. Lugo JN, Smith GD, Arbuckle EP, White J, Holley AJ, Floruta CM, Ahmed N, Cell Biol. 2008;28(12):3894–904. Gomez MC, Okonkwo O. Deletion of PTEN produces autism-like behavioral 38. Nishiyama M, Skoultchi AI, Nakayama KI. Histone H1 recruitment by CHD8 is deficits and alterations in synaptic proteins. Front Mol Neurosci. 2014;7:27. essential for suppression of the Wnt-β-catenin signaling pathway. Mol Cell 61. Dubruc E, Putoux A, Labalme A, Rougeot C, Sanlaville D, Edery P. A new Biol. 2012;32(2):501–12. intellectual disability syndrome caused by CTNNB1 haploinsufficiency. Am J 39. Wang P, Lin M, Pedrosa E, Hrabovsky A, Zhang Z, Guo W, Lachman HM, Med Genet A. 2014;164A(6):1571–5. Zheng D. CRISPR/Cas9-mediated heterozygous knockout of the autism 62. Kuechler A, Willemsen MH, Albrecht B, Bacino CA, Bartholomew DW, van gene CHD8 and characterization of its transcriptional networks in Bokhoven H, van den Boogaard MJ, Bramswig N, Buttner C, Cremer K, et al. neurodevelopment. Mol Autism. 2015;6:55. De novo mutations in beta-catenin (CTNNB1) appear to be a frequent cause 40. Merner N, Forgeot d’Arc B, Bell SC, Maussion G, Peng H, Gauthier J, Crapper of intellectual disability: expanding the mutational and clinical spectrum. L, Hamdan FF, Michaud JL, Mottron L, et al. A de novo frameshift mutation Hum Genet. 2015;134(1):97–109. in chromodomain helicase DNA-binding domain 8 (CHD8): a case report 63. Iossifov I, O’Roak BJ, Sanders SJ, Ronemus M, Krumm N, Levy D, Stessman and literature review. Am J Med Genet A. 2016;170(5):1225–35. HA, Witherspoon KT, Vives L, Patterson KE, et al. The contribution of de Kwan et al. Journal of Neurodevelopmental Disorders (2016) 8:45 Page 9 of 10

novo coding mutations to autism spectrum disorder. Nature. 2014; progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. 515(7526):216–21. Cell. 2009;136(6):1017–31. 64. De Rubeis S, He X, Goldberg AP, Poultney CS, Samocha K, Cicek AE, Kou Y, 86. Singh KK, Ge X, Mao Y, Drane L, Meletis K, Samuels BA, Tsai LH. Dixdc1 is a Liu L, Fromer M, Walker S, et al. Synaptic, transcriptional and chromatin critical regulator of DISC1 and embryonic cortical development. Neuron. genes disrupted in autism. Nature. 2014;515(7526):209–15. 2010;67(1):33–48. 65. Snijders Blok L, Madsen E, Juusola J, Gilissen C, Baralle D, Reijnders MR, 87. Boccitto M, Doshi S, Newton IP, Nathke I, Neve R, Dong F, Mao Y, Zhai J, Venselaar H, Helsmoortel C, Cho MT, Hoischen A, et al. Mutations in DDX3X Zhang L, Kalb R. Opposing actions of the synapse-associated protein of 97- are a common cause of unexplained intellectual disability with gender- kDa molecular weight (SAP97) and Disrupted in Schizophrenia 1 (DISC1) on specific effects on Wnt signaling. Am J Hum Genet. 2015;97(2):343–52. Wnt/β-catenin signaling. Neuroscience. 2016;326:22–30. 66. Cruciat CM, Dolde C, de Groot RE, Ohkawara B, Reinhard C, Korswagen HC, 88. Srikanth P, Han K, Callahan DG, Makovkina E, Muratore CR, Lalli MA, Zhou H, Niehrs C. RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Boyd JD, Kosik KS, Selkoe DJ, et al. Genomic DISC1disruption in hiPSCs alters Wnt-β-catenin signaling. Science. 2013;339(6126):1436–41. Wnt signaling and neural cell fate. Cell Rep. 2015;12(9):1414–29. 67. Lijam N, Paylor R, McDonald MP, Crawley JN, Deng CX, Herrup K, Stevens KE, 89. Singh KK, De Rienzo G, Drane L, Mao Y, Flood Z, Madison J, Ferreira M, Maccaferri G, McBain CJ, Sussman DJ, et al. Social interaction and sensorimotor Bergen S, King C, Sklar P, et al. Common DISC1 polymorphisms disrupt Wnt/ gating abnormalities in mice lacking Dvl1. Cell. 1997;90(5):895–905. GSK3beta signaling and brain development. Neuron. 2011;72(4):545–58. 68. Long JM, LaPorte P, Paylor R, Wynshaw-Boris A. Expanded characterization 90. Seshadri S, Faust T, Ishizuka K, Delevich K, Chung Y, Kim SH, Cowles M, Niwa of the social interaction abnormalities in mice lacking Dvl1. Genes Brain M, Jaaro-Peled H, Tomoda T, et al. Interneuronal DISC1 regulates NRG1- Behav. 2004;3(1):51–62. ErbB4 signalling and excitatory-inhibitory synapse formation in the mature 69. Belinson H, Nakatani J, Babineau BA, Birnbaum RY, Ellegood J, Bershteyn M, cortex. Nat Commun. 2015;6:10118. McEvilly RJ, Long JM, Willert K, Klein OD, et al. Prenatal beta-catenin/Brn2/ 91. Clapcote SJ, Lipina TV, Millar JK, Mackie S, Christie S, Ogawa F, Lerch JP, Tbr2 transcriptional cascade regulates adult social and stereotypic Trimble K, Uchiyama M, Sakuraba Y, et al. Behavioral phenotypes of Disc1 behaviors. Mol Psychiatry. 2016;21:1417–33. missense mutations in mice. Neuron. 2007;54(3):387–402. 70. Meijer L, Flajolet M, Greengard P. Pharmacological inhibitors of glycogen 92. Abazyan B, Nomura J, Kannan G, Ishizuka K, Tamashiro KL, Nucifora F, synthase kinase 3. Trends Pharmacol Sci. 2004;25(9):471–80. Pogorelov V, Ladenheim B, Yang C, Krasnova IN, et al. Prenatal interaction of 71. O’Brien WT, Harper AD, Jove F, Woodgett JR, Maretto S, Piccolo S, Klein PS. mutant DISC1 and immune activation produces adult psychopathology. Biol Glycogen synthase kinase-3β haploinsufficiency mimics the behavioral and Psychiatry. 2010;68(12):1172–81. molecular effects of lithium. J Neurosci. 2004;24(30):6791–8. 93. Furukubo-Tokunaga K, Kurita K, Honjo K, Pandey H, Ando T, Takayama K, 72. Latapy C, Rioux V, Guitton MJ, Beaulieu JM. Selective deletion of forebrain Arai Y, Mochizuki H, Ando M, Kamiya A, et al. DISC1 causes associative glycogen synthase kinase 3β reveals a central role in serotonin-sensitive memory and neurodevelopmental defects in fruit flies. Mol Psychiatry. 2016. anxiety and social behaviour. Philos Trans R Soc Lond B Biol Sci. 2012; 94. Saito A, Taniguchi Y, Rannals MD, Merfeld EB, Ballinger MD, Koga M, Ohtani 367(1601):2460–74. Y, Gurley DA, Sedlak TW, Cross A, et al. Early postnatal GABA receptor 73. Bhakar AL, Dolen G, Bear MF. The pathophysiology of fragile X (and what it modulation reverses deficits in neuronal maturation in a conditional teaches us about synapses). Annu Rev Neurosci. 2012;35:417–43. neurodevelopmental mouse model of DISC1. Mol Psychiatry. 2016. 74. Bear MF, Huber KM, Warren ST. The mGluR theory of fragile X mental 95. Durak O, de Anda FC, Singh KK, Leussis MP, Petryshen TL, Sklar P, Tsai LH. retardation. Trends Neurosci. 2004;27(7):370–7. -G regulates neurogenesis and Wnt signaling by altering the 75. Comery TA, Harris JB, Willems PJ, Oostra BA, Irwin SA, Weiler IJ, Greenough subcellular localization of β-catenin. Mol Psychiatry. 2015;20(3):388–97. WT. Abnormal dendritic spines in fragile X knockout mice: maturation and 96. Iqbal Z, Vandeweyer G, van der Voet M, Waryah AM, Zahoor MY, Besseling JA, pruning deficits. Proc Natl Acad Sci U S A. 1997;94(10):5401–4. Roca LT, Vulto-van Silfhout AT, Nijhof B, Kramer JM, et al. Homozygous and 76. Mines MA, Yuskaitis CJ, King MK, Beurel E, Jope RS. GSK3 influences social heterozygous disruptions of ANK3: at the crossroads of neurodevelopmental preference and anxiety-related behaviors during social interaction in a and psychiatric disorders. Hum Mol Genet. 2013;22(10):1960–70. mouse model of fragile X syndrome and autism. PLoS One. 2010;5(3):e9706. 97. Smith KR, Kopeikina KJ, Fawcett-Patel JM, Leaderbrand K, Gao R, Schurmann 77. Franklin AV, King MK, Palomo V, Martinez A, McMahon LL, Jope RS. Glycogen B, Myczek K, Radulovic J, Swanson GT, Penzes P. Psychiatric risk factor ANK3/ synthase kinase-3 inhibitors reverse deficits in long-term potentiation and ankyrin-G nanodomains regulate the structure and function of cognition in fragile X mice. Biol Psychiatry. 2014;75(3):198–206. glutamatergic synapses. Neuron. 2014;84(2):399–415. 78. Mines MA, Jope RS. Glycogen synthase kinase-3: a promising therapeutic 98. Leussis MP, Berry-Scott EM, Saito M, Jhuang H, de Haan G, Alkan O, Luce CJ, target for fragile x syndrome. Front Mol Neurosci. 2011;4:35. Madison JM, Sklar P, Serre T, et al. The ANK3 bipolar disorder gene regulates 79. Yuskaitis CJ, Mines MA, King MK, Sweatt JD, Miller CA, Jope RS. Lithium psychiatric-related behaviors that are modulated by lithium and stress. Biol ameliorates altered glycogen synthase kinase-3 and behavior in a mouse Psychiatry. 2013;73(7):683–90. model of fragile X syndrome. Biochem Pharmacol. 2010;79(4):632–46. 99. Singh KK. Dixdc1 is a critical regulator of DISC1 and embryonic cortical 80. Min WW, Yuskaitis CJ, Yan Q, Sikorski C, Chen S, Jope RS, Bauchwitz RP. development supplemental information. Neuron. 2010. Elevated glycogen synthase kinase-3 activity in Fragile X mice: key 100. Kivimae S, Martin PM, Kapfhamer D, Ruan Y, Heberlein U, Rubenstein JL, metabolic regulator with evidence for treatment potential. Cheyette BN. Abnormal behavior in mice mutant for the Disc1 binding Neuropharmacology. 2009;56(2):463–72. partner, Dixdc1. Transl Psychiatry. 2011;1, e43. 81. Chen X, Sun W, Pan Y, Yang Q, Cao K, Zhang J, Zhang Y, Chen M, Chen F, 101. Martin PM, Stanley RE, Ross AP, Freitas AE, Moyer CE, Brumback AC, Iafrati J, Huang Y, et al. Lithium ameliorates open-field and elevated plus maze Stapornwongkul KS, Dominguez S, Kivimae S, et al. DIXDC1 contributes to behaviors, and brain phospho-glycogen synthase kinase 3-beta expression psychiatric susceptibility by regulating dendritic spine and glutamatergic in fragile X syndrome model mice. Neurosciences. 2013;18(4):356–62. synapse density via GSK3 and Wnt/beta-catenin signaling. Mol Psychiatry. 2016. 82. Guo W, Murthy AC, Zhang L, Johnson EB, Schaller EG, Allan AM, Zhao X. Epub ahead of print. Inhibition of GSK3β improves -dependent learning and 102. Kwan V, Meka DP, White SH, Hung CL, Holzapfel NT, Walker S, Murtaza N, rescues neurogenesis in a mouse model of fragile X syndrome. Hum Mol Unda BK, Schwanke B, Yuen RK, Habing K, Milsom C, Hope KJ, Truant R, Genet. 2012;21(3):681–91. Scherer SW, Calderon de Anda F, Singh KK. DIXDC1 Phosphorylation and 83. Matic K, Eninger T, Bardoni B, Davidovic L, Macek B. Quantitative Control of Dendritic Morphology Are Impaired by Rare Genetic Variants. Cell phosphoproteomics of murine Fmr1-KO cell lines provides new insights Rep. 2016 Nov 8;17(7):1892-1904. doi: 10.1016/j.celrep.2016.10.047. into FMRP-dependent mechanisms. J Proteome Res. 103. Zhou XL, Giacobini M, Anderlid BM, Anckarsater H, Omrani D, Gillberg C, 2014;13(10):4388–97. Nordenskjold M, Lindblom A. Association of adenomatous polyposis coli 84. Luo Y, Shan G, Guo W, Smrt RD, Johnson EB, Li X, Pfeiffer RL, Szulwach KE, (APC) gene polymorphisms with autism spectrum disorder (ASD). Am J Med Duan R, Barkho BZ, et al. Fragile x mental retardation protein regulates Genet B Neuropsychiatr Genet. 2007;144B(3):351–4. proliferation and differentiation of adult neural stem/progenitor cells. PLoS 104. Sowers LP, Loo L, Wu Y, Campbell E, Ulrich JD, Wu S, Paemka L, Wassink T, Genet. 2010;6(4), e1000898. Meyer K, Bing X, et al. Disruption of the non-canonical Wnt gene PRICKLE2 85. Mao Y, Ge X, Frank CL, Madison JM, Koehler AN, Doud MK, Tassa C, Berry leads to autism-like behaviors with evidence for hippocampal synaptic EM, Soda T, Singh KK, et al. Disrupted in schizophrenia 1 regulates neuronal dysfunction. Mol Psychiatry. 2013;18(10):1077–89. Kwan et al. Journal of Neurodevelopmental Disorders (2016) 8:45 Page 10 of 10

105. Sowers LP, Mouw TJ, Ferguson PJ, Wemmie JA, Mohapatra DP, Bassuk AG. The non-canonical Wnt ligand Wnt5a rescues morphological deficits in Prickle2-deficient hippocampal neurons. Mol Psychiatry. 2013;18(10):1049. 106. Nagaoka T, Tabuchi K, Kishi M. PDZ interaction of Vangl2 links PSD-95 and Prickle2 but plays only a limited role in the synaptic localisation of Vangl2. Scientific reports. 2015;5:12916. 107. Martin PM, Yang X, Robin N, Lam E, Rabinowitz JS, Erdman CA, Quinn J, Weiss LA, Hamilton SP, Kwok PY, et al. A rare WNT1 missense variant overrepresented in ASD leads to increased Wnt signal pathway activation. Transl Psychiatry. 2013;3, e301. 108. Christensen J, Gronborg TK, Sorensen MJ, Schendel D, Parner ET, Pedersen LH, Vestergaard M. Prenatal valproate exposure and risk of autism spectrum disorders and childhood autism. Jama. 2013;309(16):1696–703. 109. Hall AC, Brennan A, Goold RG, Cleverley K, Lucas FR, Gordon-Weeks PR, Salinas PC. Valproate regulates GSK-3-mediated axonal remodeling and synapsin I clustering in developing neurons. Mol Cell Neurosci. 2002;20(2):257–70. 110. Wang L, Liu Y, Li S, Long ZY, Wu YM. Wnt signaling pathway participates in valproic acid-induced neuronal differentiation of neural stem cells. Int J Clin Exp Pathol. 2015;8(1):578–85. 111. Wiltse J. Mode of action: inhibition of histone deacetylase, altering WNT- dependent gene expression, and regulation of beta- catenin—developmental effects of valproic acid. Crit Rev Toxicol. 2005;35(8– 9):727–38. 112. Zhang Y, Sun Y, Wang F, Wang Z, Peng Y, Li R. Downregulating the canonical Wnt/β-catenin signaling pathway attenuates the susceptibility to autism-like phenotypes by decreasing oxidative stress. Neurochem Res. 2012;37(7):1409–19. 113. Go HS, Kim KC, Choi CS, Jeon SJ, Kwon KJ, Han SH, Lee J, Cheong JH, Ryu JH, Kim CH, et al. Prenatal exposure to valproic acid increases the neural progenitor cell pool and induces macrocephaly in rat brain via a mechanism involving the GSK-3β/β-catenin pathway. Neuropharmacology. 2012;63(6):1028–41. 114. Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, Klein PS. Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem. 2001;276(39):36734–41. 115. Fang WQ, Chen WW, Jiang L, Liu K, Yung WH, Fu AK, Ip NY. Overproduction of upper-layer neurons in the neocortex leads to autism-like features in mice. Cell Rep. 2014;9(5):1635–43. 116. Emamian ES, Hall D, Birnbaum MJ, Karayiorgou M, Gogos JA. Convergent evidence for impaired AKT1-GSK3beta signaling in schizophrenia. Nat Genet. 2004;36(2):131–7. 117. Sutton LP, Honardoust D, Mouyal J, Rajakumar N, Rushlow WJ. Activation of the canonical Wnt pathway by the antipsychotics haloperidol and clozapine involves dishevelled-3. J Neurochem. 2007;102(1):153–69. 118. Sutton LP, Rushlow WJ. The effects of neuropsychiatric drugs on glycogen synthase kinase-3 signaling. Neuroscience. 2011;199:116–24. 119. Launay JM, Mouillet-Richard S, Baudry A, Pietri M, Kellermann O. Raphe- mediated signals control the hippocampal response to SRI antidepressants via miR-16. Transl Psychiatry. 2011;1:e56. 120. Baudry A, Mouillet-Richard S, Schneider B, Launay JM, Kellermann O. miR-16 targets the serotonin transporter: a new facet for adaptive responses to antidepressants. Science. 2010;329(5998):1537–41. 121. Klein PS, Melton DA. A molecular mechanism for the effect of lithium on development. Proc Natl Acad Sci U S A. 1996;93(16):8455–9. 122. Hedgepeth CM, Conrad LJ, Zhang J, Huang HC, Lee VM, Klein PS. Activation of the Wnt signaling pathway: a molecular mechanism for lithium action. Dev Biol. 1997;185(1):82–91. 123. Zhang F, Phiel CJ, Spece L, Gurvich N, Klein PS. Inhibitory phosphorylation Submit your next manuscript to BioMed Central of glycogen synthase kinase-3 (GSK-3) in response to lithium. Evidence for autoregulation of GSK-3. J Biol Chem. 2003;278(35):33067–77. and we will help you at every step: 124. Mines MA, Jope RS. Brain region differences in regulation of Akt and GSK3 by • We accept pre-submission inquiries chronic stimulant administration in mice. Cell Signal. 2012;24(7):1398–405. 125. Mines MA, Beurel E, Jope RS. Examination of methylphenidate-mediated • Our selector tool helps you to find the most relevant journal behavior regulation by glycogen synthase kinase-3 in mice. Eur J • We provide round the clock customer support Pharmacol. 2013;698(1–3):252–8. • Convenient online submission 126. Kahn M. Can we safely target the WNT pathway? Nat Rev Drug Discov. 2014;13(7):513–32. • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research

Submit your manuscript at www.biomedcentral.com/submit